Redox Sensing by Fe2+ in Bacterial Fur Family Metalloregulators

Abstract

Significance: Iron is required for growth and is often redox active under cytosolic conditions. As a result of its facile redox chemistry, iron homeostasis is intricately involved with oxidative stress. Bacterial adaptation to iron limitation and oxidative stress often involves ferric uptake regulator (Fur) proteins: a diverse set of divalent cation-dependent, DNA-binding proteins that vary widely in both metal selectivity and sensitivity to metal-catalyzed oxidation. Recent Advances: Bacteria contain two Fur family metalloregulators that use ferrous iron (Fe²⁺) as their cofactor, Fur and PerR. Fur functions to regulate iron homeostasis in response to changes in intracellular levels of Fe²⁺. PerR also binds Fe²⁺, which enables metal-catalyzed protein oxidation as a mechanism for sensing hydrogen peroxide (H₂O₂).

Critical issues: To effectively regulate iron homeostasis, Fur has an Fe²⁺ affinity tuned to monitor the labile iron pool of the cell and may be under selective pressure to minimize iron oxidation, which would otherwise lead to an inappropriate increase in iron uptake under oxidative stress conditions. Conversely, Fe²⁺ is bound more tightly to PerR but exhibits high H₂O₂ reactivity, which enables a rapid induction of peroxide stress genes.

Future directions: The features that determine the disparate reactivity of these proteins with oxidants are still poorly understood. A controlled, comparative analysis of the affinities of Fur/PerR proteins for their metal cofactors and their rate of reactivity with H₂O₂, combined with structure/function analyses, will be needed to define the molecular mechanisms that have facilitated this divergence of function between these two paralogous regulators.

Keywords: Bacillus subtilis; Staphylococcus aureus; hydrogen peroxide; iron; metalloregulation; oxidative stress.

FIG. 1.

Iron-related stress responses in Bacillus subtilis. Schematic summary of the response mechanisms in B. subtilis to different types of iron-related stresses. When cells are subjected to iron starvation, cells express genes involved in iron acquisition, which encode iron importers and siderophore biosynthesis enzymes. Cells also express the small RNA FsrA, which targets post-transcriptional inhibition of low-priority iron requiring enzymes, thus implementing the iron sparing response. Under conditions of oxidative stress, cells induce expression of peroxide detoxifying enzymes (KatA and AhpC) and their cofactors (HemAXCDBL), as well as enzymes that have an impact on cellular iron levels, such as the iron sequestration ferritin-like protein (MrgA) and the P_1B4-type ATPase iron efflux pump PfeT. Note that under iron overload conditions, PfeT and (to a lesser extent) MrgA also confer resistance to excess iron. AhpC, alkyl hydroperoxide reductase; Fe, iron; KatA, catalase.

FIG. 2.

Fur and PerR structures. (A) Fur_Bs structural homology model based on the Streptomyces coelicolor Zur structure (PDB: 3mwm; sequence identity: 33.3%) (102). This Fur_Bs model shows the predicted homodimer in its holo-form. Close-up views of its three conserved metal-binding sites are provided in stick form. Site 1 is a Cys-rich pocket that binds a structural Zn²⁺ atom to provide protein stability to the dimer. Sites 2 and 3 comprise the iron-sensing site (shown without metal atoms), where site 2 seems to play a primary role in triggering the conformational changes required for DNA binding [figure adapted from (78)]. (B) PerR_Bs crystal structure bound with Mn²⁺. A close-up of the metal-sensing site (site 2) and the structural site (site 1) is depicted in stick form (PDB code: 3F8N). (C) Crystal structure of Fur_Mg:Zn,Mn (PDB code: 4RAZ) showing a close-up of binding site 1, which is analogous to site 2 in Fur_Bs and in PerR_Bs. Mn²⁺ is not easily accessible. (D) Crystal structure of PerR_Bs:Zn,Mn (PDB code: 3F8N) showing a close-up of binding site 2. Mn²⁺ is easily accessible. (E) Crystal structure of PerR_Bs:Zn,Mn showing a close-up of binding site 2 with conserved Asp104 residue mutated into a Glu. Mn²⁺ is completely occluded (Mn²⁺ = yellow; nitrogen atom = blue; oxygen atom = red; carbon atom = green). Asp, aspartate; Cys, cysteine; Fur, ferric uptake regulator; Glu, glutamate; His, histidine; Mn, manganese; Zn, zinc.

FIG. 3.

Mechanism of gene regulation by PerR_Bs. (A) Apo-PerR (PerR:Zn) lacks a metal cofactor at its metal-sensing site. This causes PerR to adopt a conformation that prevents it from binding to its DNA operator sites. As a result, no gene repression takes place. (B) As Mn²⁺ concentrations increase, apo-PerR becomes metallated, triggering a conformational change in PerR:Zn,Mn allowing it to bind its specific operator sites and repress PerR-regulated genes. Note that PerR:Zn,Mn is insensitive to oxidizing agents. (C) As Fe²⁺ concentrations increase, apo-PerR becomes metallated by iron. PerR:Zn,Fe binds to PerR operator sites and repress genes in the PerR regulon. However, during aerobic growth or in the presence of ROS, the ability of PerR:Zn,Fe to act as a repressor is limited by protein oxidation. (D) Oxidation of PerR exposes a conserved signature residue sequence recognized by LonA. Thus, oxo-PerR is targeted for degradation by the protease, preventing accumulation of inactive protein. (E) Metallated PerR can reversibly bind to Mn²⁺ or Fe²⁺ as metal concentrations in the cell vary. (F) Overview of the ability of PerR to repress various genes as a function of metal availability. In the presence of iron, under aerobic conditions, inactivation of PerR prevents accumulation of high levels of active repressor. As a result, metallated PerR:Zn,Fe is most efficient at repression of genes postulated to have high-affinity operator sites (katA, mrgA, pfeT). PerR:Zn,Mn, which is not susceptible to oxidation, can accumulate to a high effective level and strongly represses genes the entire PerR regulon, including those genes postulated to have the lowest affinity operator sites (perR, fur) (as highlighted by box). ROS, reactive oxygen species.

FIG. 4.

Metal-catalyzed oxidation of PerR. Formation of 2-oxo-histidine of PerR_Bs:Zn,Fe site 2 via three putative pathways. His37 or His91 can both become oxidized when exposed to H₂O₂. This can lead to either (A) the generation of a hydroxyl radical via oxidation of Fe²⁺ into Fe³⁺ as a result of the homolytic cleavage of the O-O of the peroxido intermediate or (B) the production of a high valent Fe⁴⁺ ion (especially under pH ∼7.0) caused by the heterolytic cleavage of that same bond. Conversely, (C) in the presence of O₂, His37 is specifically targeted, resulting in a superoxo-Fe³⁺ intermediate. Subsequent bond formation and O-O bond cleavage of the end derivative leads to the final production of 2-oxo-His [figure adapted from (100)].

FIG. 5.

Different iron-binding Fur family regulators display different sensitivities to H₂O₂. Variation in H₂O₂ sensitivity among Fur family metalloregulators. The key difference seems to lie in the structural variations between Fur and PerR at the metal-sensing site. However, other differences also likely affect H₂O₂ sensitivity. PerR_Sa:Zn,Fe is more sensitive to oxidation by H₂O₂ than PerR_Bs:Zn,Fe, and Fur_Ec seems to be more sensitive to peroxides than Fur_Bs, although the basis for these differences has not been completely defined.